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Afadin: A key molecule essential for structural organization of cell-cell junctions of polarized epithelia during embryogenesis.

Ikeda W, Nakanishi H, Miyoshi J, Mandai K, Ishizaki H, Tanaka M, Togawa A, Takahashi K, Nishioka H, Yoshida H, Mizoguchi A, Nishikawa S, Takai Y - J. Cell Biol. (1999)

Bottom Line: Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm.Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies.These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Osaka 565-0871, Japan.

ABSTRACT
Afadin is an actin filament-binding protein that binds to nectin, an immunoglobulin-like cell adhesion molecule, and is colocalized with nectin at cadherin-based cell-cell adherens junctions (AJs). To explore the function of afadin in cell-cell adhesion during embryogenesis, we generated afadin(-/-) mice and embryonic stem cells. In wild-type mice at embryonic days 6.5-8.5, afadin was highly expressed in the embryonic ectoderm and the mesoderm, but hardly detected in the extraembryonic regions such as the visceral endoderm. Afadin(-/-) mice showed developmental defects at stages during and after gastrulation, including disorganization of the ectoderm, impaired migration of the mesoderm, and loss of somites and other structures derived from both the ectoderm and the mesoderm. Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm. Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies. These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.

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Targeted disruption of the afadin gene. (A) Restriction maps of the wild-type allele, the targeting vector, and the targeted allele of the afadin gene. Filled boxes, exons. S, SacI; H, HindIII; Sp, SphI; X, XbaI; P1, PCR primer, 5′-TTCTAGGATTTGGAGTTTCAT-3′; and P2, PCR primer, 5′-GGTCAGGACACAGTCTTCACT-3′. (B) Southern blot analysis of ES clones. HindIII-digested DNAs derived from ES cells were hybridized with the 5′ or 3′ probe. (C) Southern blot analysis of mice. The afadin+/− mice were intercrossed. HindIII-digested DNAs derived from tails of the progeny were hybridized with the the 3′ probe. +/+, wild type; and +/−, heterozygous mutant.
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Figure 2: Targeted disruption of the afadin gene. (A) Restriction maps of the wild-type allele, the targeting vector, and the targeted allele of the afadin gene. Filled boxes, exons. S, SacI; H, HindIII; Sp, SphI; X, XbaI; P1, PCR primer, 5′-TTCTAGGATTTGGAGTTTCAT-3′; and P2, PCR primer, 5′-GGTCAGGACACAGTCTTCACT-3′. (B) Southern blot analysis of ES clones. HindIII-digested DNAs derived from ES cells were hybridized with the 5′ or 3′ probe. (C) Southern blot analysis of mice. The afadin+/− mice were intercrossed. HindIII-digested DNAs derived from tails of the progeny were hybridized with the the 3′ probe. +/+, wild type; and +/−, heterozygous mutant.

Mentions: A SacI-SphI genomic fragment (4.6 kb) 5′ to the afadin exon 2 encoding amino acids (aa) 36–100 was blunt-ended and inserted into the SmaI site of pBluescript neo/DT-A, which contained neomycin-resistance and diphtheria toxin A genes under the control of the MC1 promoter (Thomas and Capecchi 1987; Yagi et al. 1990). The XbaI genomic fragment (5.3 kb) 3′ to exon 2 was then inserted into the EcoRV site of pBluescript neo/DT-A containing the 5′ genomic fragment. The ∼1.0 kb SphI-XbaI fragment was targeted for disruption and replaced by the neomycin-resistant gene cassette (see Fig. 2). This fragment began at the SphI site within exon 2 and ended in the following intron, and contained the coding sequence of aa 85–100. In the targeting vector, a stop codon resided 36 bp 3′ from the encoded aa 84. For Southern blot analysis, a 0.9-kb SacI-HindIII fragment and a 1.0-kb XbaI fragment were used as 5′ and 3′ probes, respectively.


Afadin: A key molecule essential for structural organization of cell-cell junctions of polarized epithelia during embryogenesis.

Ikeda W, Nakanishi H, Miyoshi J, Mandai K, Ishizaki H, Tanaka M, Togawa A, Takahashi K, Nishioka H, Yoshida H, Mizoguchi A, Nishikawa S, Takai Y - J. Cell Biol. (1999)

Targeted disruption of the afadin gene. (A) Restriction maps of the wild-type allele, the targeting vector, and the targeted allele of the afadin gene. Filled boxes, exons. S, SacI; H, HindIII; Sp, SphI; X, XbaI; P1, PCR primer, 5′-TTCTAGGATTTGGAGTTTCAT-3′; and P2, PCR primer, 5′-GGTCAGGACACAGTCTTCACT-3′. (B) Southern blot analysis of ES clones. HindIII-digested DNAs derived from ES cells were hybridized with the 5′ or 3′ probe. (C) Southern blot analysis of mice. The afadin+/− mice were intercrossed. HindIII-digested DNAs derived from tails of the progeny were hybridized with the the 3′ probe. +/+, wild type; and +/−, heterozygous mutant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169488&req=5

Figure 2: Targeted disruption of the afadin gene. (A) Restriction maps of the wild-type allele, the targeting vector, and the targeted allele of the afadin gene. Filled boxes, exons. S, SacI; H, HindIII; Sp, SphI; X, XbaI; P1, PCR primer, 5′-TTCTAGGATTTGGAGTTTCAT-3′; and P2, PCR primer, 5′-GGTCAGGACACAGTCTTCACT-3′. (B) Southern blot analysis of ES clones. HindIII-digested DNAs derived from ES cells were hybridized with the 5′ or 3′ probe. (C) Southern blot analysis of mice. The afadin+/− mice were intercrossed. HindIII-digested DNAs derived from tails of the progeny were hybridized with the the 3′ probe. +/+, wild type; and +/−, heterozygous mutant.
Mentions: A SacI-SphI genomic fragment (4.6 kb) 5′ to the afadin exon 2 encoding amino acids (aa) 36–100 was blunt-ended and inserted into the SmaI site of pBluescript neo/DT-A, which contained neomycin-resistance and diphtheria toxin A genes under the control of the MC1 promoter (Thomas and Capecchi 1987; Yagi et al. 1990). The XbaI genomic fragment (5.3 kb) 3′ to exon 2 was then inserted into the EcoRV site of pBluescript neo/DT-A containing the 5′ genomic fragment. The ∼1.0 kb SphI-XbaI fragment was targeted for disruption and replaced by the neomycin-resistant gene cassette (see Fig. 2). This fragment began at the SphI site within exon 2 and ended in the following intron, and contained the coding sequence of aa 85–100. In the targeting vector, a stop codon resided 36 bp 3′ from the encoded aa 84. For Southern blot analysis, a 0.9-kb SacI-HindIII fragment and a 1.0-kb XbaI fragment were used as 5′ and 3′ probes, respectively.

Bottom Line: Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm.Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies.These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Osaka 565-0871, Japan.

ABSTRACT
Afadin is an actin filament-binding protein that binds to nectin, an immunoglobulin-like cell adhesion molecule, and is colocalized with nectin at cadherin-based cell-cell adherens junctions (AJs). To explore the function of afadin in cell-cell adhesion during embryogenesis, we generated afadin(-/-) mice and embryonic stem cells. In wild-type mice at embryonic days 6.5-8.5, afadin was highly expressed in the embryonic ectoderm and the mesoderm, but hardly detected in the extraembryonic regions such as the visceral endoderm. Afadin(-/-) mice showed developmental defects at stages during and after gastrulation, including disorganization of the ectoderm, impaired migration of the mesoderm, and loss of somites and other structures derived from both the ectoderm and the mesoderm. Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm. Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies. These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.

Show MeSH
Related in: MedlinePlus